The overall objective of the proposed research is to develop and test a model for the role of the signal sequence in protein secretion, particularly in the initial encounter between the nascent protein and membrane components. The importance of signal sequence conformation and membrane-binding properties will be investigated through the study of synthetic signal peptides. The sequences of these signal peptides will be chosen from secreted proteins of E. coli in which mutations have been selected or created within the signal region. The in vivo functions of these mutants will be correlated with the conformational preferences and membrane interactions of their signal peptides. As a model is developed for the role of the signal peptide in the secretion process, signal sequence mutants will be designed to test the model in vivo and via the physical properties of the isolated peptides. Methods to be used to study the conformations of the signal peptides include circular dichroism, nuclear magnetic resonance, infrared spectroscopy, and X-ray diffraction. A range of environments will be examined: bulk solvents, micelles, small unilamellar vesicles, or multilamellar vesicles. The membrane-binding properties of the signal peptides will be studied by tensiometry at air-water and lipid-water interfaces and by vesicle binding/fusion assays. Results of the proposed research should shed light on critical aspects of protein secretion both in prokaryotes and eukaryotes, and should also provide insight into membrane-peptide interactions in general.